p-menth-1-en-8-ol

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 November 2018 – 10 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 3.2, 100 and 320 mg/L.
- Sampling method: 3 test concentrations along with the negative control were analyzed for the test item concentration at the beginning and at the end of the test. One test flask (1*100 mL) for each concentration was used for chemical analysis.An aliquot was sampled at the middle of the flask.
- Sample storage conditions before analysis: All the solutions were kept in refrigerator.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was soluble in the test medium (OECD TG 201 medium) with a mixing protocol carried out before the experimentation, according to the OECD nº23 guidance document on aquatic toxicity testing of difficult substances and mixtures.
The method consisted in stirring each test item nominal concentrations in test medium during 24 hours, in the dark and 20±2ºC prepared by adding directly the test item in flasks containing test medium. For technical feasibility issues, the concentration 3.2 mg/L was diluted from the concentration 320 mg/L. After this step, a settling period of 30 minutes was performed before sampling.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: green alga Pseudokirchneriella subcapitata
- Strain: chlorococcal order strain number CCAP 278.4
- Source (laboratory, culture collection): Not reported.
- Method of cultivation: Not reported.

ACCLIMATION
- Acclimation period: Not reported.
- Culturing media and conditions (same as test or not): The algae cultures were stored in the laboratory on a solid medium (LC/agar Petri plates) and in a liquid medium with regular sub-culturing (every week).
- Any deformed or abnormal cells observed: Not reported.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test temperature:

22.7ºC - 23.1ºC

pH:

7.6 - 8.2

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 3.2, 10, 32, 100 and 320 mg/L.
Measured concentrations for 0, 3.2, 100 and 320 mg/L (initial time, T0h): <1 (control), 2.16, 78.6 and 250 mg/L.
Measured concentrations for 0, 3.2, 100 and 320 mg/L (final time, T72h): <1 (control), 2.57, 77.5 and 257 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: erlenmeyer flask
- Material, size, headspace, fill volume: glass, 250 mL capacity, 100 mL fill volume.
- Aeration: Not specified.
- Initial cells density: 10E+04 cells/mL
- Control end cells density: 168.8 x 10E+04 cells/mL (average)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes (OECD TG 201 medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the OECD medium was prepared as per the OECD guideline 201.
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH of the controls and the test solutions was checked at the beginning and at the end of the test. Temperature inside the algal chamber was recorded twice daily.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Constant illumination
- Light intensity and quality: 5924 Lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: Beckman Coulter Z2 and Malassez counting cell (observation times: 24, 48 and 72 hours)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: geometric factor of 3.2
- Justification for using less concentrations than requested by guideline: Not applicable; 5 concentrations used for the main test.

- Range finding study
- Test concentrations: 0 (control), 1, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: At the tested concentrations of 1, 10 and 100 mg/L, the average inhibition growth rate was calculated to be 6.6%, 5.3% and 45.1% respectively.

Reference substance (positive control):

yes

Remarks:

(potassium dichromate)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

124 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Confidence interval = [113 – 127]

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Confidence interval = [20 – 24]

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

41 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Confidence interval = [34 – 48]

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Lowest tested concentration (= 1.90 mg/L by calculation, for information)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on significant difference compared to control observed at the concentration 10 mg/L (Statistical significance determined with the Dunnett test (α=5%))

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Not reported.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not reported.

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the last algal growth inhibition test performed with the reference item K2Cr2O7 (June 11, 2018) indicates that the algae sensitivity is correct (within the accepted interval of 0.65 to 1.73 mg/L fixed by the International Standard NF EN ISO 8692 from Mai 2012 as mentioned in the OECD 201 Guideline) and in accordance with previous data obtained by the laboratory.
- ErC50 (0-72h) = 1.12 mg/L (1.04 – 1.20)

Reported statistics and error estimates:

For NOEC determination, statistical significance was determined with the Dunnett test (α=5%) from raw data with the scientific software Minitab 17.

Table 1: pH values during the test

Nominal test item concentration (mg/L)	pH
	At the start	After 72 hours
Control	8.2	7.6
3.2	8.1	7.8
10	8.1	7.7
32	8.1	7.7
100	8.2	7.7
320	8.2	7.7

Table 2. Algal growth inhibition

Nominal test item concentration		[algae] *104 /mL at 24 hours			[algae] *104 /mL at 48 hours			[algae] *104 /mL at 72 hours
		Count.	Average	CV	Count.	Average	CV	Count.	Average	CV
control	a	3,8	3,3	15,0	19,7	22,4	21,3	189,2	168,8	6,6
	b	2,8			24,1			168,8
	c	3,8			30,0			171,2
	d	2,9			24,5			158,6
	e	3,5			18,3			160,3
	f*	2,8			17,5			164,4
3,2	a	2,9	3.1	10.3	17,7	19.6	14.5	167,0	167.0	5.9
	b	3,5			22,9			176,8
	c	3,0			18,3			157,2
10	a	3,2	3.1	5.6	22,7	20.1	12.2	126,5	133.7	5.3
	b	3,2			19,9			133,8
	c	2,9			17,8			140,8
32	a	2,8	2.8	0.0	16,6	15.2	8.7	81,9	76.3	6.3
	b	2,8			14,0			73,6
	c	2,8			14,9			73,5
100	a	2,2	2.2	6.8	6,9	6.6	8.8	19,3	18.7	3.8
	b	2,1			5,9			18,8
	c	2,4			6,9			17,9
320	a	2,5	2.5	2.3	2,8	2.7	14.8	3,4	3.0	13.3
	b	2,5			2,3			2,6
	c	2,4			3,1			3,1

Table 3. Coefficients of variation for section-by-section specific growth rates in the control cultures

Control replicates		Specific growth rate
		0-24h	Average	CV (%)	24-48h	Average	CV (%)	48-72h	Average	CV (%)	0-72h	Average	CV per replicate (%)	Average CV
Control	a	1,335	1,174	12,7	1,646	1,914	12,2	2,262	2,038	10,6	1,748	1,709	27,0	29,9
	b	1,030			2,153			1,947			1,710		35,0
	c	1,335			2,066			1,742			1,714		21,4
	d	1,065			2,134			1,868			1,689		33,0
	e	1,253			1,654			2,170			1,692		27,2
	f	1,030			1,833			2,240			1,701		36,2

Table 4. Inhibitions of the average specific growth rate in comparison to the Control

Nominal test item concentration		algae *104 /mL at 0 hours	Specific growth rate μ0-72h	Inhibition growth rate (% Ir)	Average inhibition of growth rate (%)	Coefficient of variation	Significance
Control	a	1	1,748	Not applicable		1.2	-
	b		1,710
	c		1,714
	d		1,689
	e		1,692
	f*		1,701
3.2	a	1	1,706	0,2	0,2	Not required	ns
	b		1,725	-0,9
	c		1,686	1,4
10	a	1	1,613	5,6	4,5		*
	b		1,632	4,5
	c		1,649	3,5
32	a	1	1,468	14,1	15,5		*
	b		1,433	16,2
	c		1,432	16,2
100	a	1	0,987	42,3	42,9		*
	b		0,978	42,8
	c		0,962	43,7
320	a	1	0,408	76,1	78,4		*
	b		0,319	81,4
	c		0,377	77,9

- : not relevant

ns : not significantly different compared to the Control. Dunnet test, α =0.05

* significantly different compared to the Control, Dunnett test, α=0.05

The equation of the regression (linear regression) is the following (rounded values):

% inhibition growth rate = - 0.9247 + 0.5280 [Test item concentration] - 0.000875 [Test item concentration]2

Table 5. Inhibitions of the yield in comparison to the Control

Nominal test item concentration		[algae] *104 /mL at 0 hours	[algae] *104 /mL at 72 hours	Yield  (*104/mL)	Average yield  (*104/mL)	Yield inhibition (Iy) (%)
						Per replicate	Average/  replicate
Control	a	1	189,2	188,2	167,8	Not Applicable
	b		168,8	167,8
	c		171,2	170,2
	d		158,6	157,6
	e		160,3	159,3
	f*		164,4	163,4
3.2	a	1	167,0	166,0	166,0	1,0	1,0
	b		176,8	175,8		-4,8
	c		157,2	156,2		6,9
10	a	1	126,5	125,5	132,7	25,2	20,9
	b		133,8	132,8		20,8
	c		140,8	139,8		16,7
32	a	1	81,9	80,9	75,3	51,8	55,1
	b		73,6	72,6		56,7
	c		73,5	72,5		56,8
100	a	1	19,3	18,3	17,7	89,1	89,5
	b		18,8	17,8		89,4
	c		17,9	16,9		89,9
320	a	1	3,4	2,4	2,0	98,6	98,8
	b		2,6	1,6		99,0
	c		3,1	2,1		98,7

The equation of the regression (linear regression) is the following (rounded values):

% Yield inhibition = 7.846 + 1.138 [Test item concentration] - 0.002673 [Test item concentration]2

Table 6: Conclusion on the chemical analysis

	Concentration (mg test item / L)		Deviation (%)
Nominal test item concentration (mg/L)	Initial time (T0h)	Final time (T72h)
control	<1	<1	NA
3.2	2.16	2.57	+19.0
100	78.6	77.5	-1.4
320	250	257	+2.8

According to the results, the measured concentrations of test item in samples were maintained within ± 20 per cent of the measured initial concentration. Consequently, the test item “P-MENTH-1-EN-8-OL was considered as stable in the testing conditions after 72 hours of exposure. Therefore, and in agreement with the guideline, concentrations were expressed in nominal test item concentration.

Validity criteria fulfilled:

yes

Remarks:

(increase of biomass in control during 72h>16 fold; coefficient of variation of the mean specific growth rate among replicates in control (t0-t72)<7%; the mean of the replicate coefficients of variation in section-by-section growth rate in control < 35%)

Conclusions:

In an Alga Growth Inhibition Test with Pseudokirchneriella subcapitata green algae, the growth rate inhibition effect concentrations were determined to be 72 h-ErC50 = 124 mg/L and 72 h-ErC10 = 22 mg/L.

Executive summary:

An Algae Growth Inhibition Test was performed with Pseudokirchneriella subcapitata green algae on the test substance over a period of 72 h in static conditions according to OECD Guideline 201, following GLP. OECD medium was used for algal culturing. In the previous solubility study the test item was found soluble in OECD medium and thus no vehicle was needed. In a preliminary range finding study the average growth inhibition rate were 6.6%, 5.3% and 45.1% at the tested concentrations of 1, 10 and 100 mg/L, respectively. Based on these results, the definitive test was conducted with nominal test item concentrations of 0 (control), 3.2, 10, 32, 100 and 320 mg/L (factor of 3.2). Negative control consisted of test medium alone and potassium dichromate was used as reference substance.Three replicates were performed for each concentration of the test item, along with six replicates for the negative control group. A validated analytical method based on GC/FID was used to monitor the concentration and stability of the active ingredient in the test solution. The test item was considered as stable in the testing conditions after 72 hours of exposure as the measured concentrations in samples were maintained within ± 20 per cent of the measured initial concentration. All validity criteria were fulfilled. The percent inhibitions in average specific growth rate were 0.2, 4.5, 15.5, 42.9 and 78.4% and the percent inhibitions in yield were 1.0, 20.9, 55.1, 89.5 and 98.8% at the tested concentrations of 3.2, 10, 32, 100 and 320 mg/L respectively, when compared to the negative control. Based on these results, the growth rate inhibition effect concentrations were determined to be 72 h-ErC10 = 22 mg/L and 72 h-ErC50 = 124 mg/L, and the yield inhibition effect concentrations were 72h-EyC10 < 3.2 mg/L and 72h-EyC50 = 41 mg/L.

Description of key information

Key study. Test method according to OECD 201, GLP study. The growth rate inhibition effect concentrations to Pseudokirchneriella subcapitata green algae were determined to be 72 h-ErC50 = 124 mg/L and 72 h-ErC10 = 22 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

124 mg/L

EC10 or NOEC for freshwater algae:

22 mg/L

Additional information

Key study: An Algae Growth Inhibition Test was performed with Pseudokirchneriellasubcapitata green algae on the test substance over a period of 72 h in static conditions according to OECD Guideline 201, following GLP. OECD medium was used for algal culturing. In the previous solubility study the test item was found soluble in OECD medium and thus no vehicle was needed. In a preliminary range finding study the average growth inhibition rate were 6.6%, 5.3% and 45.1% at the tested concentrations of 1, 10 and 100 mg/L, respectively. Based on these results, the definitive test was conducted with nominal test item concentrations of 0 (control), 3.2, 10, 32, 100 and 320 mg/L (factor of 3.2). Negative control consisted of test medium alone and potassium dichromate was used as reference substance.Three replicates were performed for each concentration of the test item, along with six replicates for the negative control group. A validated analytical method based on GC/FID was used to monitor the concentration and stability of the active ingredient in the test solution. The test item was considered as stable in the testing conditions after 72 hours of exposure as the measured concentrations in samples were maintained within ± 20 per cent of the measured initial concentration. All validity criteria were fulfilled. The percent inhibitions in average specific growth rate were 0.2, 4.5, 15.5, 42.9 and 78.4% and the percent inhibitions in yield were 1.0, 20.9, 55.1, 89.5 and 98.8% at the tested concentrations of 3.2, 10, 32, 100 and 320 mg/L respectively, when compared to the negative control. Based on these results, the growth rate inhibition effect concentrations were determined to be 72 h-ErC10 = 22 mg/L and 72 h-ErC50 = 124 mg/L, and the yield inhibition effect concentrations were 72h-EyC10 < 3.2 mg/L and 72h-EyC50 = 41 mg/L.